Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation.

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Development and characterization of monoclonal antibodies against canine PD-1 and PD-L1.

Recent research has focused on immunotherapy, particularly with regard to cancer treatment. Programmed death-1 and programmed death ligand 1 (PD-1/PD-L1) pathway blockade is a central topic of the promising immunotherapy field. In veterinary medicine, observations of the PD-1/PD-L1 pathway, including the relationship between immune cells and diseases, have increased. In this study, monoclonal antibodies specific to canine PD-1 and PD-L1 were developed, and the antibodies against PD-1 and PD-L1 bind to PD-1 and PD-L1 overexpressing cells, respectively. Additionally, each antibody interfered with the interaction between PD-1 and PD-L1. The expression of PD-1 and PD-L1 was detected on activated T cells from canine peripheral blood mononuclear cells (PBMC), and, remarkably, was the first recorded instance of PD-L1 expression on canine immature dendritic cells. Production of IFN-γ by activated T cells increased significantly when incubated with anti-PD-1 antibody alone and with both anti-PD-1 and anti-PD-L1 antibodies, revealing the functional effects of the antibodies. The antibodies will be useful for research on immune systems and may be the first passive immunotherapy approach in canine cancer patients.

Programmed Cell Death Ligand 1 Expression in Canine Cancer.

BACKGROUND: Antibody therapy targeting programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a promising therapy in human cancer, but only limited information on PD-L1 expression in canine tumors is available. MATERIALS AND METHODS: PD-L1 expression was examined in 31 canine tumor cell lines of various origins by flow cytometry and western blotting, and in canine tumor and normal tissue specimens by immunohistochemistry. RESULTS: PD-L1 was only expressed on the cell surface of a small number of cell lines but was found expressed within the cells of almost all cell lines. Immunohistochemistry revealed that PD-L1 is frequently expressed in malignant melanoma, mammary gland tumor, mast cell tumor and lymphoma, but less frequently in soft-tissue sarcoma and hemangiosarcoma. PD-L1 was also expressed in some of the cells of normal canine tissue specimens. CONCLUSION: Canine tumors with PD-L1 expression that were identified in this study are potential candidates for antiPD-1 and antiPD-L1 therapy.